Optical detection of particles in a liquid medium

ABSTRACT

Particles in a liquid medium are detected by directing a source of laser light through a container containing a liquid sample with a concentration of microscopic particles in suspension. An optical detector located off the optical axis receives scattered light from the particles and provides a signal with a dc component and a varying component. The DC component is removed to provide a filtered signal representing the varying component. Movement is produced in the sample liquid, e.g., by convective stirring, to extend the band of frequencies of the varying component towards higher frequencies. The apparatus can be used for growth curve detection of biological samples and provides increased sensitivity.

[0001] The present invention relates to optical apparatus for detectingparticles in a liquid medium and is particularly concerned with thedetection of bacteria and a method of growth curve analysis of liquidbiological samples using optical techniques.

[0002] Optical techniques have been used for some years for thedetection and classification of particles in liquids. The concentrationof particles in a liquid can be measured using a turbidimeter. A beam oflight is transmitted through a liquid sample and attenuation of thelight received at a detector provides a measure of the concentration ofparticles in the sample. Reference is made to Blackburn C de W, et al,(1987) “Brief Evaluation of a Fully Automated Optical Analyser System,the Bioscreen for Measuring the Growth of Micro-organism”. TechnicalNotes No. 57, Leatherhead Food R. A.

[0003] It is also known to monitor light scattered from particles in aliquid sample. The total amount of light scattered from an illuminatedvolume of sample and received at a detector can provide an indication ofthe concentration of scattering centres or particles in the sample. Thistechnique is called nephelometry and has been used for immunoassayreaction detection.

[0004] Techniques have also been described using coherent light from alaser and monitoring the speckle pattern produced in the light scatteredfrom particles in the sample. Both the effect of interference betweenlight from different scattering centres in the sample due to differentpath lengths to the detector, and the effect of small Doppler changes inwavelengths of scattered light due to particle motion, have beeninvestigated. U.S. Pat. No. 4,826,319 and U.S. Pat. No. 6,011,621describe measuring or monitoring the size of particles in a liquid byobserving the frequency content of the scattered light.

[0005] It has also been proposed to detect living microorganisms inliquid samples by observing the characteristic frequencies of intensityfluctuations of scattered light-resulting from the motility of theorganisms. See, for example, “Spectral Analysis of Laser Light Scatteredfrom Motile Micro-organisms”, by R. Nossal, Biophysical Journal Vol. 11(1971), pp341-354. However, there are substantial technical problems inusing such techniques for practical purposes, especially for sampleswith low concentrations of micro-organisms or other particulates.

[0006] For the theory and a mathematical analysis of coherent lightscattering from particles suspended in a liquid, reference should bemade to “Photon Correlation and Light Beating Spectroscopy” edited by H.Z. Cummins and E. R. Pike, Plenum Press, 1974, especially pages ?-?.

[0007] The present invention provides optical apparatus for detectingparticles in a liquid medium, comprising a container for a liquid samplewith a concentration of microscopic particles in suspension, a source ofcoherent light arranged to direct coherent light along a predeterminedoptical axis through a sample in said container to provide anilluminated volume of said sample, a detector located off said opticalaxis and arranged to receive light from said source which has beenscattered by particles in said illuminated volume of said sample, saiddetector providing a signal representing the intensity of said receivedlight and comprising a dc component dependent on the concentration ofsaid particles in said illuminated volume and a time varying componentwith frequencies in a band, a filter removing said dc component toprovide a filtered output signal, and a sample stirring device toproduce movement of the sample liquid in said illuminated volume so asto extend said band of frequencies towards higher frequencies.

[0008] The total intensity of light received by a detector from ailluminated volume of liquid sample is dependent on, amongst otherthings, the number of scattering particles in the volume. However, thistotal intensity also contains the following additional components:

[0009] a component dependent on molecular scattering by the molecules ofthe liquid of the sample,

[0010] b) components dependent upon absorption of light by the liquidand the material of the sample container,

[0011] c) a component dependent on scattering by the material of thesample container, and

[0012] d) a component dependent on scattering and absorption in thewindow of the photo detector device.

[0013] Accordingly, the component which is representative of the amountof light being scattered from particulates (in particular from bacteriaor other micro-organisms) in the sample can be only a relatively smallproportion of the total received signal, especially for low particleconcentrations.

[0014] The present invention removes the dc component of the totalscattered intensity signal from the detector, to provide a filteredsignal representing only the variations in the total intensity. By usinga coherent light source, such as a laser, to provide the illuminatedvolume of the sample, the amplitude of the variations of the scatteredlight intensity is also representative of the number of scatteringparticles within the sample volume. Interference between light scatteredfrom individual particles within the illuminated volume produces aninterference pattern (speckle pattern) at the plane of the detector.Because the scattering particles within the illuminated volume of theliquid sample are in motion, experiencing Brownian motion at least, thespeckle pattern is continuously changing, producing the variations inthe intensity signal from the detector. The amplitude of these intensityvariations is generally proportional to the number of scattering centresor particles in the illuminated volume.

[0015] Importantly, the other sources of scattered light received by thedetector tend not to produce variations in the detected intensity. As aresult, filtering out the dc component of the detected intensity,produces a signal which can represent the concentration of scatteringparticles within the illuminated volume with much greater sensitivity.

[0016] According to theory, as set out in Cummins and Pike referencedabove, it can be shown that the spectral content of variations in theintensity of scattered light from particles moving solely with Brownianmotion, include substantial power density at very low frequencies (ofintensity variation), down to 0.1 Hz and less for particles of 1μ sizeand low scattering angles (e.g. 5-7 deg). By comparison, the energy athigher variation frequencies, say 10 Hz or more, is 1000 times less. Bystirring the sample liquid to produce movement of the liquid in theilluminated volume, the detected intensity variations are pushed towardshigher frequencies, and it becomes possible to filter out the dccomponent of the detected intensity of scattered light effectivelywithout using a filter with an excessively long time constant.

[0017] As a result, the optical apparatus described above can detectchanges in the concentration of scattering particles in a sample fluidwith great sensitivity and with a shorter response time. Importantly,where the optical apparatus is used for monitoring the growth curve ofbiological samples containing a growth medium, growth of the biologicalspecies in the particular medium can be detected much sooner and at muchlower concentrations.

[0018] Conveniently, the sample stirring device comprises a heaterarranged to heat the sample in the container to produced convectivestirring. In order to achieve the required extension of the bandwidth ofthe frequency of intensity variation, the stirring device should producea velocity of liquid movement of the order of one millimeter per second.Preferably, the stirring action of the stirring device provides liquidmotion in different directions in different parts of the illuminatedvolume of the sample. As a result, the stirring action itself producescorresponding changes in the speckle pattern formed in the plane of thedetector. It has been found that the half band or relaxation frequencyof the frequency spectrum of intensity variation of the scattered lightis substantially linearly related to the mean speed of motion of liquidin the illuminated volume of the sample caused by the stirring device. Arelatively low level of stirring action in the liquid sample canincrease the half band frequency from as low as 0.1 Hz for Brownianmotion of 1 μparticles in still liquid, to 200 to 300 Hz or more. As aresult, the filter can be set with a lower cut off frequency of about 10Hz and still pass a substantial proportion of the energy contained inthe spectrum of intensity variation for the scattered light.

[0019] As mentioned above, sufficient stirring of the liquid sample canbe achieved using a heater. Preferably, the heater comprises a heaterblock for receiving said container, the heater block being asymmetricabout a vertical plane containing the optical axis. In this way, theheater can be arranged to heat the sample differentially in a horizontalplane to ensure adequate convective stirring of the sample to producethe required bandwidth extension to higher frequencies.

[0020] In a preferred embodiment, the apparatus comprises at least afirst matched pair of said detectors equally spaced at a common radialdistance on opposite sides of a plane containing said optical axis, anddifference means receiving input signals respectively from said pair ofdetectors and providing a different signal representing the differencebetween said input signals. In this way, correlated signal variationsreceived by both detectors symmetrically spaced on opposite sides of theplane containing the optical axis are rejected in the difference signal.On the other hand, the intensity variations received by each detectorwhich are dependent on interference in the scattered light, are notcorrelated and so the amplitude of the non correlated signal variationswill be additive in the difference signal to produce a total amplitudeequal to {square root}2 times the amplitude of variation from each ofthe two detectors. In this way, the signal to noise ratio for thedesired intensity variation signal can be substantially increased.Common mode signal variations which would be characteristic of manynoise sources in the apparatus, are received equally and in phase by thetwo matched detectors of the pair and cancel each other in thedifference signal. In particular, all intensity variations resultingfrom noise in the light source should be cancelled in this way.

[0021] The apparatus may include one or more further said pairs ofdetectors. A respective difference signal is obtained from the outputsof the detectors of each pair and the various difference signals canthen be summed so as further to increase the sensitivity for detectionof variations in the scattered light resulting from changes in theinterference pattern caused by motion of the scattering particles in theilluminated volume of sample liquid.

[0022] Very preferably, the or each detector has a sensitive aperturewhich is greater than ten times the coherence area for interferingscattered light at the detection plane normal to the optical axis. Thecoherence area is a measure of the grain size of the speckle pattern atthe plane of the detector caused by interference between light scatteredby the various particles in the illuminated volume of the sample.Classical theory for the detection of intensity variations resultingfrom the speckle pattern suggests that the aperture at the detectorshould normally be in the range of 1 to 5 coherence areas (see“Biochemical Applications of Laser Rayleigh Scattering” by N. C. Ford,Jr., Chemica Scripta 1972,2,193-206). However, it has been found thatbetter signal to noise is obtained using a relatively large illuminatedvolume of sample (which would lead to a small coherence area at thedetector), in combination with a relatively large aperture of detectorembracing a substantial number of coherence areas. The overall dc levelof signal produced by the detector is then increased in proportion tothe increase in area, and it has been found that the amplitude ofvariations in the output signal are also increased in proportion to thesquare root of the increase in area.

[0023] In practice, the illuminated volume of said sample may have anarea normal to the optical axis which is greater than 1 mm².

[0024] Also, the illuminated volume may have a dimension along theoptical axis which is greater than 3 mm, and preferably greater than 7mm. Overall, the illuminated volume may be greater than 3 mm³, andpreferably greater than 30 mm³.

[0025] The sensitive detector aperture may be between 0.5 and 2 mm².

[0026] The distance between the mid point of the illuminated volumealong the optical axis and the detection plane may be between 1 and 10cm.

[0027] The present invention also provides a method of growth curveanalysis of a liquid biological sample comprising the steps of:

[0028] preparing a liquid sample for analysis containing a selectedgrowth medium,

[0029] illuminating a volume of said sample with coherent light directedalong an optical axis,

[0030] detecting the intensity of light scattered from said illuminatedvolume,

[0031] filtering out a dc component of said detected intensity toprovide a filtered signal representing only intensity variations in apredetermined band of frequencies,

[0032] monitoring said filtered signals over a period of time, and

[0033] determining from changes in said filtered signal the likelypresence or absence of a biological species which is viable in theselected growth medium.

[0034] By monitoring only the intensity variations in light reflectedfrom an illuminated sample, changes in the concentration of scatteringcentres in the sample, which will normally represent a change in thepopulation of a biological species in the sample, can be detected withmuch greater sensitivity and therefore at a much earlier stage in thedevelopment of a growth path.

[0035] Preferably, the method includes the step of producing motion ofthe liquid of said sample relative to the optical axis. As explainedabove, this shifts the frequencies of the intensity variations to higherfrequencies, allowing a greater total energy of the intensity variationsignal to be detected whilst eliminating the dc content, without usingfilters with excessive time constants.

[0036] Preferably the band of frequencies monitored is from 1 Hz to 500Hz, and more preferably between 10 Hz and 300 Hz.

[0037] Examples of the present invention will row be described withreference to the accompanying drawings, in which:

[0038]FIG. 1 is a schematic illustration of particle detection apparatusembodying the present invention;

[0039]FIG. 2 is a schematic view in elevation of a pair of cuvettescontaining liquid samples in a heating block for use in the apparatus ofFIG. 1;

[0040]FIG. 3 is a schematic plan view of the heating block of FIG. 2illustrating the heating and temperature control arrangement;

[0041]FIG. 4 is a schematic view taken along the optical axis of thelaser beam illustrating an arrangement of photodetectors which may beemployed in the apparatus of FIGS. 1 to 3;

[0042]FIG. 5 is a schematic block diagram of an electronic circuitemployed to provide an output signal from the apparatus;

[0043]FIG. 6 is a graphical representation of a typical Lorentzian powerdensity frequency spectrum of the fluctuations of intensity of lightreceived at a detector which has been scattered by particles in Brownianmotion in the illuminated volume of a liquid sample;

[0044]FIG. 7 is a corresponding power density distribution for particlesin Brownian motion where the liquid of the sample is being stirred toproduce movement of the liquid relative to the optical axis of theilluminating light beam; and

[0045]FIG. 8 is a graphical representation of a growth curve obtained bythe apparatus of FIG. 1 for a sample containing a micro-organism in agrowth medium.

[0046] Referring to FIG. 1, a beam 10 from a laser 11 is directed alongan optical axis 12 through a liquid sample 13 contained in a sampleholder or cuvette 14. The cuvette 14 is made from a material which issubstantially transparent to the laser light, or at least has atransparent window through which the laser beam can enter the cuvette toilluminate the liquid sample 13, and a further transparent windowthrough which light scattered from particles in an illuminated volume 15of the liquid can pass to a detector 16.

[0047] As illustrated, the laser beam 10 is substantially collimated sothat the illuminated region 15 of sample liquid within the cuvette 14has a volume corresponding to the product of the cross-sectional area ofthe laser beam and the width of the cuvette in the direction of theoptical axis 12. Also, in the illustrated example, scattered light fromall parts of the illuminated region 15 of the liquid sample can reachthe sensitive area of the photodetector 16.

[0048] The photodetector 16 produces on line 17 a signal representing atany time the intensity of scattered light received by the detector. Thissignal on line 17 is filtered in a dc filter 18 to remove the dccomponent of the signal so that only the variations in the receivedsignal are passed to a monitor 19.

[0049] It is important to note that, by comparison with prior artphotometric apparatus such as disclosed in U.S. Pat. Nos. 4,826,319 and6,011,0621, the apparatus described in this embodiment of the inventionis not concerned with detecting or analysing the spectral content ofvariations in light scattered from the liquid sample. In this example ofthe invention, it is the total energy in these signal variations whichis being detected and monitored.

[0050] Furthermore, it should be understood that the describedembodiment is particularly concerned with the analysis of liquid sampleswith relatively low concentrations of scattering particles.

[0051] As mentioned before, the amplitude of variations in the scatteredsignal detected by the detector 16 has a generally proportionalrelationship to the concentration of scattering centres. However, inpractice, the detector 16 must have a dynamic range sufficient toaccommodate the dc level of scattered light from the large illuminatedvolume 15, and still sufficiently low noise to permit the much smallerfluctuation amplitude still to be detected.

[0052] The described apparatus may be compared with nephelometry inwhich the intensity of scattered light is monitored, but instead thedescribed apparatus uses a coherent light source and filters out the dclevel of the scattered light intensity signal so as to use thefluctuation amplitude as the measuring parameter for particleconcentration in the sample.

[0053] The laser 11 may be a solid state semiconductor laser operatingat 670 nm. The wavelength is not however critical for many applications.The laser 11 produces a collimated beam having a cross-sectional areagreater than about 1 mm², and typically having a diameter of about 3 mm.The width of the interior of the cuvette 14 in the direction of theoptical axis 12 is normally greater than 3 mm and may be about 1 cm. Asa result, the illuminated region 15 may have a volume of about 70 mm².

[0054] To maximise the amount of scattered light which can be receivedby the detector 16, the detector is located off the optical axis 12, butas close as possible to the beam 10 without receiving any directillumination from the beam. Further, the detector 16 is located close tothe cuvette 14 to increase the amount of scattered light received by thedetector. The sensitive area of the detector 16 may be about 1 mm² andit may be located at about 2 cms from the nearer wall of the cuvette 14,so that the line between the centre of the illuminated region 15 and thecentre of the detector 16 forms an angle of between 5° and 7° with theoptical axis 12 of the laser beam.

[0055] Referring now to FIG. 6, according to the theory which is knownto the those skilled in this art, the power density spectrum ofintensity fluctuations of the scattered light has a Lorentz distributionof the form illustrated in the Figure, on the assumption that thescattering particles are moving only with Brownian motion. Forscattering particles having a size of about 1μ and a scattering angle ofabout 5°, the relaxation frequency or half band frequency of thespectrum is at about 0.1 Hz. It can be seen, therefore, that a verysubstantial part of the power in the intensity fluctuations (as much as99%) is at frequencies below 1 Hz, and as much as 50% is below 0.1 Hz.

[0056] The embodiment of the apparatus illustrated in FIG. 1, includes astirrer 20 illustrated adjacent a lower end of the cuvette 14, which iseffective to produce a stirring motion of the liquid sample in thecuvette.

[0057] Again, the mechanism which produces the stirring action is not initself critical. Some mechanical agitation of the liquid sample couldwork, although it is desirable to avoid movement of the cuvette 14itself relative to the ion beam 10.

[0058] In a preferred embodiment, the stirrer 20 comprises a heateradapted to produce convective currents in the fluid sample.

[0059] The motion of the sample liquid 13 produced by the stirrer 20 hasan important effect on the power density spectrum of intensityvariations In the scattered-light received by the detector 16. Asillustrated in FIG. 7, the movement of the sample liquid has the effectof extending the spectrum towards higher frequencies. Thus, curve 22 inFIG. 7 represents the power density spectrum from light scattered byparticles in the sample liquid when the sample liquid is stirred to havea first typical speed relative to the ion beam 10 (say about 0.5 mm/s),and curve 23 represents the power density spectrum of the same sample(with the same concentration of scattering particles) but for a higherspeed of movement of the sample (say about 1 mm/s). As can be seen, asthe velocity of movement is increased, the relaxation frequency of thespectrum moves to higher frequency values with a simultaneous reductionin the constant power density level for frequencies below the relaxationfrequency. In fact, it has been found that the relaxation frequency ofthe power density spectrum is substantially proportional to the velocityof movement of the liquid sample.

[0060] In the example illustrated in FIG. 7, the speed of liquid motionof about 1 millimetre per second produces a relaxation frequency ofaround 200 Hz.

[0061] Importantly, the increase in the relaxation frequency of thepower density spectrum produced by the circulation or stirring motion ofthe liquid sample allows the dc filter 18 to operate with a low pass cutoff of about 10 Hz. As a result, the dc filter 18 can be made to respondwith a relatively shorter time constant, whilst still responding to amajor portion of the total power in the intensity variation signal.

[0062] Typically, the filter 18 will also be arranged to have an upperfrequency cut off to eliminate higher frequency noise and otherdisturbing elements from the signal. So long as the higher frequency cutoff is substantially higher than the relaxation frequency, the resultingfiltered signal is relatively insensitive to changes in the speed ofmotion of the stirred liquid sample in the cuvette 14. As can be seenfrom FIG. 7, the total power in the filtered signal is represented bythe area under the respective curve 22 or 23 and this area does notchange significantly as the relaxation frequency increases due to thecompensating reduction in the constant level at lower frequencies.

[0063] Accordingly, the stirrer 20 provides an important enhancement ofthe sensitivity of the apparatus disclosed in FIG. 1 which also allowsthe filtered signal from the filter 18 to respond more quickly tochanges in the concentration of scattering particles in the illuminatedvolume 15 of the sample.

[0064] Importantly also, the stirring effect of the liquid samplereduces any tendency for particles to settle out and ensures that thetotal number of particles in the illuminated volume is a fairrepresentation of the overall concentration of particles in the wholesample 13. Also, when the apparatus is used for performing a microbiological assay, such as a growth curve analysis, the stirring effectof the sample improves response by ensuring any viable micro biologicalparticles are continually exposed to food from the growth mediumcontained in the sample. This is done without the need for agitation ofthe cuvette 14 which could have an effect on the filtered signal fromthe filter 18, due to changes in intensity in the light scattered fromthe walls of the cuvette for example.

[0065] A preferred form of heater for the apparatus of FIG. 1 isillustrated in FIG. 2. In FIG. 2, two cuvettes 30 and 31 are mounted ona single heating block 32. The heating block which is also illustratedin plan view in FIG. 3, has a heating element 33 and a temperaturesensor 34 providing a feedback signal to a temperature controller 35which is designed to power the heating element 33 so as to maintain theblock 32 at a predetermined temperature.

[0066] The heating block 32 is arranged to support cuvettes 30 and 31side by side in a vertical plane which is perpendicular to the opticalaxes of a pair of parallel laser beams 36 and 37. Each of the laserbeams 36 and 37 can be generated from a respective separate laser whichis not shown in these drawings. The laser beams 36 and 37 are arrangedto pass through the respective cuvettes 30 and 31 at a point below thelevels 38,39 of liquid samples in the respective cuvettes, to provideilluminated regions of the respective samples in the manner asillustrated in FIG. 1.

[0067] Each of the cuvettes 30 and 31 has a rectangular horizontalcross-section. The heater block 32 is shaped substantially as a letter Ewith a central part 40 extending vertically between and snugly fittingagainst the inner facing surfaces 41 and 42 of the two cuvettes. Thecentral part 40 extends up the height of the cuvettes to above the levelof the laser beams 36 and 37 substantially to the levels 38 and 39 ofthe liquid samples in the cuvettes.

[0068] Outer parts 43 and 44 of the E of the heater block 32 extendvertically outside the outer faces 45 and 46 of the two cuvettes. Theouter parts 43 and 44 may be spaced from the outer surfaces 45 and 46 ofthe cuvettes, as shown, and also do not extend to the same height as thecentral part 40. In fact in FIG. 2, the outer parts 43 and 44 do notextend as high as the level of the laser beams 36 and 37, but this isnot critical.

[0069] As illustrated in FIG. 3, the heater block 32 is substantiallythe same width, in the laser beam direction, as the cuvettes 30 and 31at the plane of the laser beams, and the front and back faces of thecuvettes are exposed to receive the laser beam on one side and to allowscattered light to emerge for detection on the opposite side of therespective cuvettes.

[0070] With the asymmetric construction of the heater block 32illustrated in FIG. 2, a sample in each of the respective cuvettes 30and 31 is heated asymmetrically so that there will be a slighttemperature difference between the inner face of the cuvette adjacentthe central part 40 of the heater block, and the outer faces of thecuvettes which are generally exposed to the local ambient atmosphere. Asa result, a convective current will flow in the liquid sample asillustrated by the arrow 47.

[0071] It has been found that a sufficient movement of the sample toachieve the desired objective of shifting the relaxation frequency ofthe power density spectrum to higher frequencies (FIG. 7) can beobtained with only a relatively small temperature difference across thesample. For example, with cuvettes 30 and 31 having a substantiallysquare cross-section at the level of laser beams 36 and 37 of side about1 cm, only a very small temperature difference across the liquid sample(typically about 0.1° C.) is sufficient to ensure adequate stirringmotion of the liquid. Such a temperature differential can be assuredwith the difference between the temperature of the sample liquid withinthe cuvette and ambient in the vicinity of the outer faces of thecuvette, of just 3° C. This may readily be achieved when operating theheater 33 to maintain the heater block and the liquid samples in thecuvettes, substantially at a temperature of about 37° C. which is astandard temperature for promoting bacterial growth in a growth medium.

[0072] Referring now to FIG. 4, this is a schematic illustration of aphotodetector arrangement which may be used for detecting scatteredlight in embodiments of the present invention.. The illustration of FIG.4 is a view along the optical axis 50 of a laser beam 51 taken at apoint substantially in the plane of the photodetectors such asphotodetector 16 in FIG. 1. Instead of a single photodetector 16 asillustrated in FIG. 1, the arrangement of FIG. 4 provides twelveseparate detector elements in groups of four which are mounted togetherin units 52, 53 and 54. Each of the units 52, 53 and 54 are similar.Considering unit 52, this contains four individual photodetectorelements 56 to 58 each having sensitive surfaces about 1 mm². Apreferred form of photosensitive element is the photodiode type QD7-5from RS Components. The four photodiodes 55 to 58 are mounted in twopairs 55,56 and 57,58 with the diodes of each pair being mountedsymmetrically on either side of a plane 59 containing the optical axis50 of the laser beam 51. Thus, the two photodiodes of each symmetricalpair should receive the same dc component of intensity of scatteredlight. Similarly, the diodes of the pair should provide intensityvariation signals having similar amplitude and frequency distribution.However, the intensity variation components of the signals will beuncorrelated with respect to each other.

[0073] In the arrangement illustrated, each of the blocks 52, 53 and 54contain four diodes mounted together as illustrated so that there are atotal of six pairs of symmetrically positioned diodes.

[0074] Referring now to FIG. 5, the signals from each pair ofsymmetrically positioned diodes are supplied to the inverting and noninverting inputs respectively of a respective differential amplifier.Thus, the signals from photodiodes 55 and 56 are supplied via respectivedc blocking capacitors 57 and 58 to a differential amplifier 59.Similarly, signals from diodes 60 and 61 are supplied via capacitors 62and 63 to differential amplifier 64. The signals from the other pairs ofdiodes as illustrated in FIG. 4 are also supplied to correspondingdifferential amplifiers but these have been represented by dashed linesin FIG. 5 for simplicity.

[0075] The difference signals from the six differential amplifiers arethen supplied to a summing amplifier 65 and the resulting sum signal issupplied to a band pass filter 66. The output of the band pass filter isA/D converted in ADC 67 and the resulting digital information providedto a computer 68.

[0076] By providing signals from symmetrically positioned and matchedpairs of photodiodes to the inputs of respective differentialamplifiers, any correlated varying components of the two signals fromthe two diodes of the pair will tend to cancel out in the output signalfrom the differential amplifier. Such correlated signals could forexample correspond to variations in the intensity of the laser lightfrom the laser 11. On the other hand, the intensity variations resultingfrom diffraction between light scattered by the different particles inthe liquid sample are uncorrelated with each other so that subtractingone signal from the other in the differential amplifier 59, for example,in fact produces an intensity variation signal having an amplitude whichis {square root}2 times the amplitude of each of the intensityvariations signals from the individual diodes. Accordingly, thedescribed apparatus can significantly reduce interfering components fromthe intensity variation signal whilst increasing the required component.

[0077] Similarly, the output from the summing amplifier 65 will have anintensity variation amplitude which is {square root}6 times the averageamplitude of the six difference signals from the differentialamplifiers, further increasing the sensitivity of the instrument.

[0078] The band pass filter 66 is arranged to pass only signals in thefrequency range between about 10 Hz and about 300 Hz and the resultingsignal supplied and digitised by the ADC 67 can be a sensitive andresponsive measure of the concentration of scattering particles in thesample in the cuvette. The ADC 67 may comprise a standard PC sound card.

[0079] The described apparatus can have a particular application ingrowth curve analysis of biological samples. Because of the highsensitivity and fast response time of the instrument, growth curves canbe categorised at a relatively early stage in their development.Considering the graphical illustration of FIG. 8, this represents thegrowth curve of a bacterium in a sample of human urine. The backgroundcount of scattering particles in the urine sample would be typically ofthe order of 10⁵ parts per millilitre (ppml). Curve A indicates thedevelopment of a growth curve as measured by the above describedinstrument following the introduction of the sample to a growth medium.For comparison, Curve B shows the growth curve detected by a knownturbidimeter for the same sample. In the illustrated example, growth ofthe bacteria in the sample can be detected by the above describedinstrument about three hours earlier even for fast growing bacteria(with a generation time of about 25 minutes). For slow growing bacteriathe time improvement can be eight hours or more. This compares extremelyfavourably with known prior art growth curve analysis techniques.

[0080] For cleaner samples, with a lower background particle count orconcentration, the development of a growth curve can be recognised evensooner.

[0081] It should be noted that advantages can be obtained in performinggrowth curve analysis on samples by monitoring intensity variations inthe light scattered from a volume of the sample illuminated withcoherent light. This can provide a more sensitive method of growth curveanalysis which allows an earlier characterisation of the sample.Accordingly, although it is preferred to perform growth curve analysiswith an instrument as described above in which the liquid sample isstirred in order to shift the relaxation frequency of the intensityvariation power density spectrum to higher frequencies, useful resultscan also be obtained in the absence of any substantial stirring,although this will require a trade off between response time of theinstrument and sensitivity.

[0082] A significant feature of the above described embodiment of theinvention is the use of photodiodes for detection of the intensity ofscattered light from the illuminated liquid sample. Prior artphotometric instruments commonly use photon counting techniques fordetecting scattered light, because the scattered light intensities ofsuch prior art instruments are extremely low. This is because only avery small illuminated volume of sample is imaged to the detector, oftenusing a large scattering angle and a narrow divergence angle, so thatthe detector will respond primarily to intensity variationscorresponding to movement of the particles themselves. In the embodimentdescribed above, a relatively large illuminated volume is “imaged” tothe detector, so that the detector can be considered to have a sensitivearea which is many times the coherence area of the speckle pattern inthe detection plane. As a result of this, the output of thephotodetector has a substantial dc component. However, because theamplitude of intensity variations in the detector signal are in factalso increased, filtering out the dc component of the detector signalcan result in an intensity variation signal which is a sensitive measureof relatively low concentrations of scattering particles in the sample.It is important, however, that the relative magnitude between the dccomponent of the detector signal and the intensity variation componentis not so great that the detector is either saturated by the dccomponent, or the intensity variation component becomes comparable tothe amplitude of the noise signal produced by the detector. In thedescribed example, the amplitude of intensity variation is typicallyfrom 0.1 to 1% of the dc component.

[0083] In the above described embodiment, a total of six pairs ofsymmetrically arranged photodiodes have been used to provide an enhancedintensity variation signal. Additional pairs of diodes may also be used.Furthermore arrays of large numbers of diodes could be used so long asthese could be connected in pairs which are symmetrical on either sideof planes containing the laser beam axis.

1. Optical apparatus for detecting particles in a liquid medium,comprising a container for a liquid sample with a concentration ofmicroscopic particles in suspension; a source of coherent light arrangedto direct coherent light along a predetermined optical axis through asample in said container to provide an illuminated volume of saidsample; a detector located off said optical axis and arranged to receivelight from said source which has been scattered by particles in saidilluminated volume of said sample, said detector providing a signalrepresenting the intensity of said received light and comprising a dccomponent dependent on the concentration of said particles in saidilluminated volume and a time varying component with frequencies in aband, a filter removing said dc component to provide a filtered outputsignal and a sample stirring device to produce movement of the sampleliquid in said illuminated volume so as to extend said band offrequencies towards higher frequencies.
 2. Apparatus as claimed in claim1, wherein said sample storing device comprises a heater arranged toheat the sample in the container to produce convective stirring. 3.Apparatus as claimed in claim 2, wherein said heater is arranged to heatthe sample differentially in a horizontal plane.
 4. Apparatus as claimedin claim 3, wherein said heater comprises a heater block for receivingsaid container, said heater block being asymmetric about a verticalplane containing said optical axis.
 5. Apparatus as claimed in anypreceding claim, including at least a first matched pair of saiddetectors equally spaced at a common radial distance on opposite sidesof a plane containing said optical axis, and difference means receivinginput signals respectively from said pair of detectors and providing adifference signal representing the difference between said inputsignals.
 6. Apparatus as claimed in claim 5, including at least onefurther said pair of detectors on opposite sides of said plane and at arespective different common radial distance from said optical axis, saiddifference means receiving input signals from said further pair ofdetectors and providing a further difference signal representing thedifference between the input signals from said further pair, and asumming means receiving said difference signals and providing a sumsignal representing the sum of said difference signals.
 7. Apparatus asclaimed in either of claim 5 or claim 6, including at least one furthersaid pair of detectors on opposite sides of said plane and diametricallyopposed to said first pair with respect to said optical axis, saiddifference means receiving said difference means receiving input signalsfrom said further pair of detectors and providing a further differencesignal representing the difference between the input signals from saidfurther pair, and a summing means receiving said difference signals andproviding a sum signal representing the sum of said difference signals.8. Apparatus as claimed in any of claims 5 to 7, including at least onefurther said pair of detectors at a common radial distance on oppositesides of a respective different plane containing said optical axis, saiddifference means receiving input signals from said further pair ofdetectors and providing a further difference signal representing thedifference between the input signals from said further pair, and asumming means receiving said difference signals and providing a sumsignal representing the sum of said difference signals.
 9. Apparatus asclaimed in any of claims 1 to 8, wherein the or each detector has asensitive aperture which is greater than ten times the coherence areafor interfering scattered light at the detection plane normal to theoptical axis.
 10. Apparatus as claimed in claim 9, wherein theilluminated volume of said sample has an area normal to the optical axiswhich is greater than 1 mm².
 11. Apparatus as claimed in either ofclaims 9 to 10, wherein said illuminated volume has a dimension alongsaid optical axis which is greater than 3 mm.
 12. Apparatus as claimedin claim 11, wherein said dimension is greater than 7 mm.
 13. Apparatusas claimed in any of claims 9 to 12, wherein said illuminated volume isgreater than 3 mm³.
 14. Apparatus as claimed in claim 13, wherein saidilluminated volume is greater than 30 mm³.
 15. Apparatus as claimed inany of claims 9 to 14, wherein said sensitive detector aperture isbetween 0.5 and 2 mm².
 16. Apparatus as claimed in any of claims 9 to15, wherein the distance between the mid point of the illuminated volumealong the optical axis and the detection plane is between 1 and 5 cm.17. A method of growth curve analysis of a liquid biological samplecomprising preparing a liquid sample for analysis containing a selectedgrowth medium, illuminating a volume of said sample with coherent lightdirected along an optical axis, detecting the intensity of lightscattered from said illuminated volume, filtering out a dc component ofsaid deleted intensity to provide a filtered signal representing onlyintensity variations in a predetermined band of frequencies monitoringsaid filtered signal over a period of time, and determining from changesin said filtered signal the likely presence or absence of a biologicalspecies which is viable in the selected growth medium.
 18. A method ofgrowth curve as claimed in claim 17, including the step of producingmotion of the liquid of said sample relative to the optical axis.
 19. Amethod of growth curve as claimed in claim 18, wherein said band offrequencies is from 1 Hz to 500 Hz.
 20. A method of growth curve asclaimed in claim 19, wherein said band of frequencies is from 10 Hz to300 Hz.